Int J Clin Exp Med 2018;11(7):7004-7017 www.ijcem.com /ISSN:1940-5901/IJCEM0069480

Original Article Identification of commonly regulated and biological pathways as potential targets in PC-3 and DU145 androgen-independent human prostate cancer cells treated with the curcumin analogue 1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one

Kamini Citalingam1, Faridah Abas2,3*, Nordin H Lajis2*, Iekhsan Othman1*, Rakesh Naidu1*

1Jeffery Cheah School of Medicine and Health Sciences, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar Sunway, Selangor, Malaysia; 2Laboratory of Natural Products, Faculty of Science, 3Department of Food Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. *Equal contributors. Received November 20, 2017; Accepted May 3, 2018; Epub July 15, 2018; Published July 30, 2018

Abstract: Diarylpentanoid [1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one] (MS17) demonstrated enhanced anti- cancer activity compared to curcumin but its effect on androgen-independent prostate cancer has not been well- studied. The present study was aimed to perform expression profiling on MS17 treated PC-3 and DU145 cells using microarray technology to identify mutually regulated genes as common targets in both androgen-independent prostate cancer cell lines as well as molecular pathways that contributes to the anticancer activity of MS17. The profiling data revealed a dose-dependent gene regulation, evident by higher fold change expression values when the treatment dose was increased by 1.5-fold in both cell lines. classification was performed on highly regulated differentially expressed genes (DEGs). Among these genes, the mutually regulated DEGs were identified as common targets in both cell lines. The mutually up-regulated DEGs included, CRYAB and DNAI2 associated with cytoskeletal organization, HSPA6 and HSPB8 with response to unfolded , MMP3 and MMP10 with proteoly- sis, CACNA1G with transporter activity, NGFR with apoptosis, CCL26 with immune response, DNAJA4 with protein folding and TRIML2 with protein ubiquitination. However, the down-regulated genes such as CTDSP1 were associ- ated with phosphatase activity, HIST1H2BF and HIST1H2AI with organization, MXD3 with regulation of transcription and TNFRSF6B with apoptosis. PC-3 and DU145 are androgen-independent prostate cancer cells with different biological properties, and identification of common targets in both cell lines could potentially be used as therapeutic targets. Pathway analysis of the DEGs in PC-3 cells demonstrated modulation of top pathways associ- ated with cell cycle checkpoint, DNA damage, and inflammatory response while in DU145 cells the pathways were associated with immune response and metabolism. Thus, the findings of the present study provide insight into the antitumor activity of MS17 and as a potential chemotherapeutic agent for androgen independent prostate cancer cells.

Keywords: Androgen-independent prostate cancer, diarylpentanoid, microarray gene expression profiling, pathway analysis

Introduction to prevent the progression of hormone sensi- tive prostate cancer cells to hormone refrac- Prostate cancer is the leading cause of cancer tory stage. Curcumin, a polyphenol active com- among men. Although in the initial stage pros- pound extracted from the rhizome of the tur- tate cancer cells responds well to androgen meric plant, Curcuma longa has known to dem- deprivation therapy, it often progresses to an onstrate potential anticancer effect against a androgen-independent phenotype that is resis- variety of carcinomas both in vitro and in vivo tant to treatment [1]. Despite available thera- [2]. Despite the success of curcumin in treating peutic options, much effort has been directed cancer, its poor pharmacokinetic characteris- towards developing novel treatment modalities tics stimulated chemists to synthesize variety Gene expression of prostate cancer cells of analogues with the hope to obtain com- the chemically purified diarylpentanoid, MS17 pounds that are more effective and with a wider [1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3- spectrum of anti-tumor activity while retaining one] was synthesized based on the method its safety profile [3-5]. Diarylpentanoids (DAPs), described previously [16], and was prepared by a group of curcumin-like analogues with 5-car- dissolving in 100% DMSO (Sigma-Aldrich, St. bon chain between its aryl rings was reported Louis, MO, USA) to a final concentration of 50 to display significant growth suppressive poten- mM. tial compared to curcumin [6]. Several studies have demonstrated anticancer activity of DAPs Treatment and total RNA isolation in a wide range of cancer cell lines [7-12]. EF- 24 [3,5-bis(2-fluorobenzylidene)-4-piperidone] Cells were seeded in triplicate wells containing a curcumin DAP exhibited potent growth inhibi- 4 × 105 cells/mL in 6-well tissue culture plates tory effects in a variety of cancer cells including and treated with 10 µM and 15 µM doses of prostate cancer at doses significantly lower MS17 for 24 hours. Control wells (untreated than curcumin [13]. Other investigators report- cells) containing media only with 0.5% DMSO ed the anti-tumorigenic effects of the structur- was also included. Following overnight incuba- ally identical DAP ca27 and Ca 37 in androgen- tion, cells were harvested and RNA was isolat- dependent and -independent prostate cancer ed using RNeasy® Mini Kit (Qiagen, Valencia, cells respectively [7, 14]. While many of these CA, USA) and purified using RNase-free DNA DAPs demonstrated an improved anticancer set according to the manufacturer’s protocol. effect compared to curcumin, the underlying Concentration and purity of the extracted RNA mechanisms that contributed to its anticancer was measured spectrophotometrically using activity in prostate cancer cells remains to be NanoDrop 1000 (Thermo Fisher Scientific, Wal- explored. tham, MA, USA). RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent Tech- In our recent study, curcumin diarylpentanoid nologies, Santa Clara, CA, USA) and scored as 1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3- RNA integrity number (RIN). Samples with RIN one (MS17) demonstrated improved dose- and score exceeding 8 indicate high RNA quality time-dependent growth inhibitory effects on and were selected for microarray analysis. androgen-independent prostate cancer cell lin- es, PC-3 and DU145 respectively at EC50 values Microarray of approximately 5.0 µM compared to curcum- in. MS17 also demonstrated significant apop- Genome-wide expression profiles of the treated totic effects at 24 hours of treatment with early and untreated samples were analyzed using apoptosis noted at 2 × EC50 (10 µM) and 3 × Agilent one-color microarray-based gene expre- EC50 (15 µM) concentrations in the treated cells ssion analysis protocols (Agilent Technologies). [15]. Hence, in the present study we performed Briefly, 100 ng quality-checked total RNA was gene expression profiling on MS17 treated PC-3 reverse transcribed using a Low Input Quick and DU145 cells using microarray technology Amp labeling kit and then transcribed to Cy3- to identify mutually regulated genes as com- labeled cRNA according to the manufacturer’s mon targets in both androgen-independent protocol. Cy3-labeled cRNA samples were hy- prostate cancer cell lines as well as molecular bridized onto whole SurePrint pathways that contributes to the anticancer G3 8X60K arrays (Agilent Technologies) using activity of MS17. Agilent’s Surehyb Chambers in an Agilent hy- Materials and methods bridization oven set at 65°C for 17 hours. Three independent replicates were performed for Cell culture and preparation of MS17 diaryl- each treatment. The slides were washed and pentanoid subsequently scanned by an Agilent G2600D SureScan Microarray Scanner (Agilent Techno- Androgen-independent metastatic human pro- logies). Images from the scanned array were state cancer cell line, PC-3 and DU145 cells analyzed using Agilent’s Feature Extraction were purchased from American Type Culture software, version 11.5.1.1 and further analyz- Collection (ATCC, Rockville, MD, USA) and cul- ed for statistical evaluations using GeneSpring tured as previously described [15]. Separately, GX version-13.0 software (Agilent Technologies).

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Data normalization and statistical analysis gen.com/ingenuity) and DEG’s from the pros- tate cancer cells were mapped to canonical Data normalization and transformation steps pathways using reference genes from the Inge- recommended by Agilent Technologies for one- nuity Knowledge Base. Using the Fishers Exact color oligonucleotide microarrays were per- Test the p-value was set at p < 0.05, canonical formed using GeneSpring GX software. The raw pathways were considered as statistically sig- signals were log transformed and normalized nificant when it passed through a threshold of using the Percentile shift normalization meth- 1.3 [calculated by -log (p-value)]. A ratio value od. Quality control on the probe sets were fil- calculating the number of genes from the data- tered based on flag and expression values to set that matched components within the path- remove probes with low signal intensities (< way was also calculated. 20.0). Statistical comparisons were performed between the experimental groups by using Real-time PCR (qRT-PCR) validation Analysis of Variance (ANOVA) with Benjamini- Hochberg false discovery rate (FDR) set as a Selected candidate genes which was previous- multiple test correction method, followed by a ly identified by microarray analysis at 24 hours, post hoc testing using Turkey’s Honestly Sig- were validated by qRT-PCR. The total RNA which nificance Difference (HSD) to identify pair-wise was used for microarray analysis were reverse significant genes. Genes that exhibited correct- transcribed to cDNA using the High-Capacity ed p-value < 0.05 and a fold change (FC) in cDNA Reverse Transcription kit according to expression greater than 2.0 (FC > 2.0 denotes the manufacturer’s specifications (Applied Bio- up-regulation; FC < -2.0 denotes down-regula- systems). The cDNA was then used as tem- tion) compared to control cells were identified plates for PCR amplification of selected genes as being differentially expressed genes (DEGs). using the TaqMan probes. A set of 6 genes were used to validate gene expression in PC-3 Gene Ontology (GO) classification analysis and DU145 cells which includes MMP3 (Hs- Specific FC cut-off values (PC-3; up-regulated 00968308_m1), MMP10 (Hs00233987_m1), DEGs = FC ≥ 18.0 while down-regulated DEGs = CCL26 (Hs00171146_m1), CTDSP1 (Hs011- FC ≤ -3.7 and DU145; up-regulated DEGs = FC 05503_m1), MXD3 (Hs00361007_m1) and ≥ 15 while down-regulated DEGs = FC ≤ -2.5) TNFRSF6B (Hs00187070_m1). GAPDH (Hs99- were applied to the list of DEGs to obtain a list 999905_m1) was used as the internal control. of top ranked DEGs that were under/over Each sample was run in duplicates in a final vol- expressed by treatment. Hierarchical cluster ume of 20 µL. The reactions were carried out TM analysis for the top ranked DEGs was perfor- using a StepOnePlus Real-time PCR system med to demonstrate the response to treatment (Applied Biosystems) and the cycling program by MS17. Gene Ontology (GO) analysis was also was set as follows: an initial PCR activation performed on the highly regulated genes using step at 48°C for 30 min followed by 95°C for 10 The Database for Annotation, Visualization and min. 40 cycles of melting at 95°C for 15 s and Integrated Discovery (DAVID) functional anno- annealing/extension at 60°C for 1 min. Rela- tation tool (htpp://david.abcc.ncifcrf.gov) ver- tive gene expression was calculated using the sion 6.7. The selected genes were categorized comparative CT method (ΔΔCT). Expression was based on GO functional category to provide reported as fold change in the level of mRNA of a description of the biological process (BP)/ treated cells relative to untreated cells, normal- molecular function (MF) and their correspond- ized to GAPDH expression value. Histograms ing GO terms were identified. and statistical analysis were performed using Graphpad Prism version 5.04 for Windows Pathway analysis (Graphpad Software, La Jolla, CA, USA).

The list of DEGs that were obtained from the Results microarray analysis which satisfied the p-value < 0.05 and FC cut-off criteria were selected Gene expression profiling and Gene Ontology for pathway analysis based on a database of (GO) classification analysis curated pathways. Gene symbols were import- ed into Ingenuity® Pathway Analysis (IPA) soft- The gene expression data identified a total of ware (IPA®; QIAGEN Redwood City, www.qia- 7429 and 4252 significant (p < 0.05) entities in

7006 Int J Clin Exp Med 2018;11(7):7004-7017 Gene expression of prostate cancer cells

Overall, it was noted that an increase in treatment dosage by 1.5-fold from 10 µM to 15 µM resulted in significant increase in expression levels by sever- al fold, proposing a dose- dependent pattern of gene regulation by MS17 in the prostate cancer cells. Ba- sed on Gene Ontology (GO) classification analysis, a to- tal of 19 and 22 functional categories were identified in PC-3 and DU145 cells, respectively. Among the to- tal of 19 functional catego- ries in PC-3 cells, cytoskel- etal organization constitut- ed the major proportion (16%) followed by cell cycle (15%), chromosome organi- zation (12%), regulation of transcription (10%) and binding activity (6%). How- ever, the rest of the func- tional categories represen- ted approximately 1-4% of the DEGs (Figure 2A). Amongst the 22 functional categories associated with DU145 cells, the largest Figure 1. Hierarchical cluster analysis of top deregulated DEGs in MS17-treated functional category was PC-3 and DU145 cells as compared to control cells. Specific cut-off values were applied to each group of genes and the hierarchical clustering was generated regulation of transcription based on the cut-off criteria. Overall, the hierarchical cluster analysis revealed (11%) followed by oxidore- significant differences between the MS17-treated and control groups in both ductase activity, binding cell lines. The regulation pattern was differentiated with 2 distinct colors; green activity, and transporter indicates up-regulation while red represents down-regulation and black corre- activity each comprised of sponds to intermediate regulation relative to control cells. 10%, while metabolic pro- cess accounts for 9%, sig- PC-3 and DU145 cells respectively. Of these, nal transduction activity for 7% and apoptosis 1386 genes in PC-3 and 987 genes in DU145 for 5% of the DEGs. However, the other func- cells were differentially expressed in response tional categories constitues between 1-4% of to 10 µM and 15 µM treatment of MS17. Among the DEGs (Figure 2B). these genes, a total of 33 up- and 56 down- regulated genes were obtained in PC-3 cells Among the total DEGs, 11 up- and 5 down-reg- (Supplementary Table 1) while 32 up- and 49 ulated genes in PC-3 and DU145 cells were down-regulated genes were identified as the identified as mutually regulated genes in both top ranked DEGs in MS17-treated DU145 cells dosages as well as common gene targets of (Supplementary Table 2). The top deregulated MS17 in both the cell lines. The selected genes genes were graphically represented by hierar- from both cell lines along with their correspond- chical clustering to view the changes in the ing functional categories are highlighted in gene expression patterns (Figure 1). Table 1. Among these 3 up- and 3 down-regu-

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Figure 2. Gene Ontology (GO) classification of the top deregulated DEGs in (A) PC-3 and (B) DU145 cells.

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Table 1. Selected differentially expressed genes (DEGs) mutually regulated following 10 µM and 15 µM treatment of MS17 in PC-3 and DU145 cells at 24 hrs GO Term/ Genbank PC-3 DU145 Gene description Gene Symbol accession FC (10 µM) FC (15 µM) p-Value FC (10 µM) FC (15 µM) p-Value Up-regulated Cytoskeletal organization CRYAB* NM_001885 Crystallin, alpha B 277.6 537.6 5.42E-04 334.9 493.9 5.46E-04 DNAI2* NM_023036 Dynein, axonemal, intermediate chain 2 24 51.9 7.84E-03 17.6 32.1 3.77E-02 Response to unfolded protein HSPA6* NM_002155 Heat shock 70 kDa protein 6 (HSP70B’) 145.2 336.7 1.09E-03 373.4 814.4 4.96E-04 HSPB8* NM_014365 Heat shock 22 kDa protein 8 41.3 88.4 1.67E-03 12 18 2.89E-02 Protein ubiquitination TRIML2* NM_173553 Tripartite motif family-like 2 30.2 88.1 6.54E-03 11.6 24.7 3.61E-02 Transporter activity CACNA1G* NM_018896 Calcium channel, voltage-dependent, T type, alpha 1G subunit 17 48.3 2.51E-03 23 60.2 3.60E-02 Proteolysis MMP3* NM_002422 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) 15.6 33.9 2.92E-03 71.6 122.8 5.83E-03 MMP10* NM_002425 Matrix metallopeptidase 10 (stromelysin 2) 9.2 20.9 6.03E-03 96.7 165.2 5.11E-03 Protein folding DNAJA4* NM_018602 DnaJ (Hsp40) homolog, subfamily A, member 4 16.3 31.7 1.55E-03 11.5 19.1 3.87E-03 Apoptosis NGFR* NM_002507 Nerve growth factor receptor 14.2 23.8 6.34E-03 31.5 53.6 1.15E-02 Immune response CCL26* NM_006072 Chemokine (C-C motif) ligand 26 13 23.1 1.51E-03 56 81.4 1.70E-03 Down-regulated Phosphatase activity CTDSP1* NM_021198 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 1 -5.8 -9.3 6.59E-04 -4.7 -5.8 6.10E-03 Chromosome organization HIST1H2AI* NM_003509 cluster 1, H2ai -3.7 -5.7 1.66E-03 -2.6 -2.8 3.15E-03 HIST1H2BF* NM_003522 Histone cluster 1, H2bf -3.5 -4.6 1.88E-03 -2.7 -3.3 2.38E-02 Apoptosis TNFRSF6B* NM_003823 Tumor necrosis factor receptor superfamily, member 6b, decoy -3.4 -4 2.10E-02 -4.5 -6.5 3.66E-03 Regulation of transcription MXD3* NM_031300 MAX dimerization protein 3 -2.6 -3.7 4.18E-03 -2 -2.7 1.02E-02 *Green represents mutually up-regulated genes while red indicates mutually down-regulated genes.

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Table 2. Top 5 Canonical pathways significantly modulated by mutually deregulated DEGs in 10 µM- and 15 µM-treated prostate cancer cells Nominal Canonical pathways *Genes p-value PC-3 Mitotic Roles of Polo-Like Kinase 1.58E-05 CCNB2*, FBXO5*, PLK1*, CDK1*, KIF11* Cell Cycle: G2/M DNA Damage Checkpoint Regulation 9.55E-05 TOP2A*, CCNB2*, PLK1*, CDK1* Granulocyte Adhesion and Diapedesis 2.29E-04 CLDN11*, MMP3*, MMP10*, CCL26* Agranulocyte Adhesion and Diapedesis 2.88E-04 CLDN11*, MMP3*, MMP10*, CCL26* ATM Signaling 2.40E-03 CCNB2*, BLM*, CDK1* DU145 Airway Pathology in Chronic Obstructive Pulmonary Disease 7.94E-03 MMP1* Zymosterol Biosynthesis 2.00E-02 TM7SF2* Guanosine Nucleotides Degradation III 2.40E-02 NT5E* Urate Biosynthesis/Inosine 5’-phosphate Degradation 3.16E-02 NT5E* Dermatan Sulfate Biosynthesis (Late Stages) 3.16E-02 CHST1* Note: Green represents mutually up-regulated genes while red indicates mutually down-regulated genes. lated genes were seletced for validation using Cycle: G2/M DNA Damage Checkpoint Regu- qRT-PCR. The mutually up-regulated genes lation was modulated by down-regulation of included, CRYAB and DNAI2 were associated TOP2A, CCNB2, PLK1, and CDK1 expression. with cytoskeletal organization, HSPA6 and HS- In addition, Granulocyte Adhesion and Diapede- PB8 with response to unfolded protein, MMP3 sis, and Agranulocyte Adhesion and Diapedesis and MMP10 with proteolysis, CACNA1G with pathways were modulated by induced expres- transporter activity, NGFR with apoptosis, CCL- sion of MMP3, MMP10, and CCL26 and down- 26 with immune response, DNAJA4 with pro- regulation of CLDN11 respectively. Conversely, tein folding and TRIML2 with protein ubiquitina- the fifth ranking pathway, ATM signaling was tion. Analysis of the mutually repressed DEGs modulated by the suppressed expression of demonstrated down-regulation of CTDSP1 CCNB2, BLM, and CDK1. associated with phosphatase activity, HIST1- H2BF and HIST1H2AI with chromosome organi- On the other hand, the two most significantly zation, MXD3 with regulation of transcription activated pathways in DU145 cells; Airway Path- and TNFRSF6B with apoptosis. ology in Chronic Obstructive Pulmonary Disease and Zymosterol Biosynthesis were associated Pathway analysis of the MS17 treated prostate with the induction of MMP1 and repression of cancer cells TM7SF2 respectively. The pathways, Guanosine Nucleotides Degradation III and Urate Biosyn- The top deregulated DEGs were mapped to thesis/Inosine 5’-phosphate Degradation was biological canonical pathways to identify and modulated by the down-regulation of NT5E understand the cellular pathways that signifi- respectively. In addition, Dermatan Sulfate Bio- cantly modulated by MS17 treated prostate synthesis (Late Stages) was associated with cancer cells. The top 5 canonical pathways of the up-regulation of CHST1 expression. PC-3 and DU145 cells that were significantly induced/activated by treatment were selected Validation of selected MS17-regulated genes for further analysis. For each pathway, the using qRT-PCR respective nominal p-value along with all the input genes are presented in Table 2. To substantiate the results from the microarray analysis, quantitative real-time PCR (qRT-PCR) In PC-3 cells, the most significantly activated was performed to validate the expression of six pathway, Mitotic Roles of Polo-Like Kinase was selected MS17 mutually expressed genes in associated with the down-regulation of CCNB2, the prostate cancer cells. The expression data FBXO5, PLK1, CDK1, and KIF11 while the sec- from real-time PCR was normalized against the ond most significantly activated pathway, Cell housekeeping gene, GAPDH and both results

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Figure 3. mRNA expression of selected genes from microarray analysis was confirmed using qRT-PCR. cDNA from MS17-treated (A) PC-3 and (B) DU145 cells from 3 independent experiments were used for validation of the gene expression obtained from microarray analysis. The selected up-regulated genes were MMP3, MMP10, and CCL26 while the down-regulated genes were CTSPD1, MXD3, and TNFRSF6B. Data was presented as log2 fold change in MS17-treated cells as compared to untreated cells. All data were normalized to the housekeeping gene, GAPDH. Re- sults show that the expression patterns obtained through real-time PCR were consistent with the microarray results.

were expressed as log2 fold change in MS17- while DU145 cells were derived from brain treated cells compared to untreated cells. metastasis and moderately metastatic with Validation confirmed up-regulation of MMP3, mutant p53. Despite the biological differences, MMP10, and CCL26 and down-regulation of we identifed mutually regulated DEGs as com- CTSPD1, MXD3, and TNFRSF6B gene expres- mon targets in both cell lines and DEGs mapped sions in both MS17-treated PC-3 and DU145 to relevant cellular pathways, and their role in cells, which were consistent with the microar- anticancer activity were discussed. ray data (Figure 3). CRYAB and HSPA6 associated with cytoskele- Discussion tal organization and response to unfolded protein respectively were the topmost over- In the present study, PC-3 and DU145 cells expressed transcripts and particularly relevant were used as a model of androgen-indepen- due to their mutual up-regulation in both MS17- dent human prostate cancer cells. PC-3 cells treated prostate cancer cells. CRYAB encodes were derived from bone metastasis with high alpha-B crystalline protein which is a heat- metastatic potential harbours wild-type p53 shock protein and molecular chaperone that

7011 Int J Clin Exp Med 2018;11(7):7004-7017 Gene expression of prostate cancer cells participates in intracellular architecture of the bule motor complex and associated with the cytoskeleton. CRYAB is a potential tumor-sup- dynein complex. It is involved in ciliogenesis pressor gene and over-expression of CRYAB and associated with cell motility [26, 27]. In suppressed nasopharyngeal carcinoma tumori- mitosis, dynein participates in chromosome genicity in vivo by affecting cell adhesion, mig- movements, spindle organization, spindle posi- ration, and interaction with tumor microenvi- tioning, and checkpoint silencing [26, 27]. ronment while its decreased expression corre- Up-regulation of DNAI2 and the cytoskeletal- lated with cancer progression [17]. Heat shock related genes by MS17 suggests a potential (HSPs) are powerful molecular chaper- role in maintaining the cytoskeletal architec- ones that play central role in the correct folding ture of the prostate cancer cells and inhibition of misfolded protein and drive damaged pro- may reduce the cytoskeletal organization and teins to endoplasmic reticulum for proteasome increase tumor cell motility. degradation in conditions of cellular oxidative stress [18]. HSPA6 codifies for the stress induc- Transcripts associated with proteolysis such as ible Hsp70B’ protein that plays a key role in MMP10 and MMP3 were significantly induced mediating cell survival during endoplasmic reti- in response to treatment. Metallopeptidases culum stress condition. Induced HSPA6 expres- (MMPs) are known as the collagenase-related sion by curcumin analogue D6 in melanoma connective tissue degrading MMPs that are cells caused cellular toxicity and endoplasmic associated with proteolytic degradation of the reticulum stress that resulted in apoptotic cell ECM during inflammation and immune response death [19]. In addition, induction of apoptosis [28]. MMPs were shown to play dual roles in in human schwannoma cell line exposed to cur- oncogenesis, either as oncogenes or as tumor- cumin treatment was associated with up-regu- suppressors. MMP10 is associated with pro- lation of Hsp70 [20]. Apart from HSPA6, HSPB8 teolytic degradation of the ECM during inflam- was also mutually up-regulated by 2.3- and 2.1- mation and immune response [29]. In contrast, fold respectively when treatment dose was depletion in MMP3 expression in squamous increased by 1.5-fold in PC-3 and DU145 cells. cell carcinoma tumors in vivo stimulated tumor- HSPB8 is silenced by aberrant DNA methyla- igenesis and was associated with leukocyte tion in a high proportion of melanoma pros- infiltration [30]. Induction of specific MMPs by tate cancer, Ewing’s sarcoma and hematologic MS17 proposes that it may participate in the malignancies. However, its expression induces proteolytic ECM degradation and host defence cell death and inhibits tumor growth in xeno- mechanism of the treated prostate cancer cells graft models suggesting HSPB8 could be a to exert its anti-cancer effect. tumor suppressor [21]. Curcumin was shown to induce heat shock response in human cervical CACNA1G and NGFR associated with transport- cancer and colorectal carcinoma cells [22-24]. er activity and apoptosis respectively, were Therefore, induction of these transcripts during mutually up-regulated in both prostate cancer endoplasmic stress may contribute to apopto- cells. CACNA1G codifies a T-type calcium chan- sis and suppress tumor formation. DNAJA4 nel protein which regulates cytosolic calcium associated with protein folding encodes for during cell proliferation and cell death. It has heat shock protein family Hsp40 which is a been reported that intracellular calcium signal- metastatic suppressor gene. DNAJA4 promotes ling plays an important role in apoptosis [31]. Apolipoprotein E (ApoE) expression, an anti- Up-regulation of CACNA1G expression in can- angiogenic and metastasis-suppressive factor cer cells induces apoptosis and anti-prolifera- in melanoma cells resulting in suppression of tive activity [32]. NGFR, a transmembrane gly- melanoma metastasis [25]. Endogenous ApoE coprotein from the neurotrophin family was and DNAJA4 were found to suppress metastat- reported to act as a tumour suppressor. Up- ic endothelial recruitment in vivo [25]. The regulation of NGFR expression in breast cancer increase expression of this transcript by MS17 cells facilitates anti-tumorigenesis, by negative- may induce apoptosis and mediate anti-meta- ly regulating cell proliferation and cancer cell static activity in the treated cells. growth [33]. Up-regulation of CACNAIG and NGFR by MS17 may suppress tumorigenesis by In addition to CRYAB, DNAI2 was also associ- inducing anti-proliferative activity and apopto- ated with cytoskeletal organization. DNAI2 is a sis in prostate cancer cells, thus inhibiting can- part of the large minus end-directed microtu- cer progression.

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TRIML2 associated with protein ubiquitination cluster proteins that are involved in chromo- is a member of a large tri-partite motif (TRIM) some segregation and alignment [41]. Over- protein family that are implicated in transcrip- expression of these transcripts in cancer tis- tional regulation, DNA damage, apoptosis, and sues has been associated with oncogenesis cancer [34]. Increased expression of TRIML2 in [41]. While information on the anti-tumorigenic colorectal cancer cells enhanced the activation role of HIST1H2AI is not well described, increa- of pro-apoptotic target genes while its down- sed HIST1H2BF expression in non-lung cancer regulation impaired apoptotic induction [35]. cells significantly correlated with gemcitabine Increased expression of TRIML2 by MS17 may drug resistance [42]. The down-regulation of potentially suppress tumorigenesis and con- these genes by MS17 may enhance sensitivity tribute toward pro-apoptotic activity in prosta- towards treatment and modulate chromosome te cancer cells. The immune response-related organization that impairs prostate cancer cell gene, CCL26 was also mutually up-regulated survival which requires further investigation. by treatment. CCL26 is a member of the CC Conversely, repression of MXD3 is associated Chemokine family that encodes for dendritic- with regulation of transcription and encodes a cell specific chemokines that are implicated in MAD family transcription factor whose expres- innate and acquired immune responses [36]. sion was up-regulated in mouse and human However its role in anti-tumorigenesis is yet to medulloblastomas while demonstrating anti- be known and requires further investigation. proliferative activity and increased apoptosis [43], suggesting its possible role as pro-apop- In contrast to the up-regulated genes, several totic factor and negative regulator of cell prolif- mutually repressed DEGs were also identified eration in the cancer cells. in both MS17 treated prostate cancer cells. TNFRSF6B associated with apoptosis is a solu- In the present study we have identified several ble decoy receptor from the TNFR super-family DEGs modulated by MS17 treated PC-3 and which plays a regulatory role in suppressing DU145 prostate cancer cells that were mapped FasL- and LIGHT-mediated cell death. TNFRS- to relevant biological canonical pathways. In F6B induction in liver carcinoma cells correlat- addition to the common genes discussed, ed with metastasis while its down-regulation DEGs associated with pathways were exclusive- resulted in decreased cancer cell proliferation, ly noted in either PC-3 or DU145 cells. The top activation of cell cycle arrest at G1/S phase, 5 canonical pathways that significantly activat- inhibition of cell migration and induction of ed in PC-3 cells were associated with cell cycle apoptosis [37]. Over-expression of TNFRSF6B checkpoint, DNA damage response and inflam- in colorectal cancer cells correlated with tu- matory response. The pathways “Mitotic Roles mor differentiation, lymph node metastasis of Polo-Like Kinase”, “Cell Cycle: G2/M DNA and overall poor patients survival [38]. In addi- Damage Checkpoint Regulation” and “ATM Sig- tion, down-regulation of TNFRSF6B expression naling” were modulated by the down-regulation in breast cancer cells inhibited cellular migra- of CCNB2 and CDK1. In addition, PLK1 was tion and invasion [39]. The down-regulation of also mapped to “Mitotic Roles of Polo-Like TNFRSF6B expression by MS17 may inhibit cell Kinase” and “Cell Cycle: G2/M DNA Damage proliferation and migration, activate cell cycle Checkpoint Regulation” pathways. All three arrest and induce apoptosis in prostate cancer transcripts are critical regulators of cell cycle cells. The nuclear phosphatase expressed by progression. Down-regulation of CCNB2 and CTDSP1 belongs to the protein of small C- CDK1 demonstrated increased apoptosis and terminal domain phosphatases that acts as induction of anti-proliferative activity in cancer a transcriptional regulator of neuronal gene cells [44, 45] while decreased PLK1 expression where it is commonly associated with the leads to cell cycle arrest, induction of apoptosis silencing of neuronal gene expression [40]. This and tumor suppression in cancer cells [46]. suggests a possible transcriptional regulatory Conversely, “Mitotic Roles of Polo-Like Kinase” role of CTDSP1 in the inhibition of prostate can- pathway was also associated with the modula- cer cell survival demonstrating the anti-tumori- tion of FBXO5 and KIF11 which plays a key role genic activity of MS17. in mitosis. Over-expression of FBXO5 promotes chromosome instability and the formation of HIST1H2BF and HIST1H2AI associated with solid cancers in vivo [47] while its down-regu- chromosome organization encode for histone lation inhibited cell proliferation [48]. Down-

7013 Int J Clin Exp Med 2018;11(7):7004-7017 Gene expression of prostate cancer cells regulation of KIF11, a kinesin protein, in cancer sion of proteins involved in epidermal differ- cells promotes apoptosis [49]. In addition to entiation [56]. Inhibition of NT5E expression in these genes, Cell Cycle: G2/M DNA Damage human breast cancer cells activated cell cycle Checkpoint Regulation” and “ATM Signaling” arrest, induced apoptosis, and suppressed tu- pathways were also modulated by the down- mor growth [57]. Due to its anti-tumorigenic regulation of TOP2A and BLM respectively. effects, the association of these transcripts in TOP2A and BLM were associated with DNA rep- relation to “Airway Pathology in Chronic Obstru- lication, and down-regulation in cancer cells ctive Pulmonary Disease”, “Zymosterol Biosyn- inhibits cell growth [50, 51]. In addition, “Granu- thesis”, “Dermatan Sulfate Biosynthesis (Late locyte Adhesion and Diapedesis” and “Agranu- Stages)”, “Guanosine Nucleotides Degradation locyte Adhesion and Diapedesis” pathways III” and “Urate Biosynthesis/Inosine 5’-phos- were associated with inflammatory response phate Degradation” pathways suggest the and modulated by up-regulation of MMP3, potential role of MS17 to repress tumor growth MMP10, CCL26, and down-regulation of CLD- and modulate cell cycle arrest as well as induc- N11 expression. The roles of MMP3, MMP10, tion of apoptosis in treated DU145 cells. and CCL26 have been described previously. Meanwhile, CLDN11 which belongs to the Although PC-3 and DU145 are androgen-inde- tetraspanin family of proteins are integral to pendent prostate cancer cells with differenet the structure and function of tight junctions. biological properties, we identified several Up-regulation of CLDN11 expression promoted genes mutually regulated by MS17 which could gastric cancer tumorigenesis but down-regula- potentially be used as therapeutic targets for tion inhibits cell growth [52]. Notably, the mod- androgen-independent prostate cancer cells. ulation of the abovementioned pathways by the These commonly regulated genes may inhibit target genes demonstrated anti-cancer role of cell proliferation, migration, metastases, and MS17 in contributing to anti-proliferative, tumor induce apoptosis in prostate cancer cells. The suppressive and apoptotic properties in treat- pathway analysis revealed genes mapped to ed PC-3 cells. the relevant biological pathways were mainly associated with cell cycle arrest, anti-prolifera- Likewise, the modulation of the top five canoni- tive activity, metabolism, or apoptotic cell death cal pathways in DU145 cells demonstrated the when treated with MS17. Thus the findings of activation of pathways associated with immune response and metabolism. The pathways, “Air- the present study provides an insight into the way Pathology in Chronic Obstructive Pulmonary antitumor activity of MS17 and as a potential Disease” and “Dermatan Sulfate Biosynthesis chemotherapeutic agent for androgen indepen- (Late Stages)” were modulated by the induced dent prostate cancer. expression of MMP1 and CHST1 respectively. Acknowledgements The exact function of individual MMPs may vary from tissue remodelling, angiogenesis, wound This study was financially supported by the healing to inflammation, but MMP1 expression Fundamental Research Grant Scheme, (FRGS/ was shown to promote tissue remodeling-asso- 1/2013/SKK01/MUSM/02/1) under the Mini- ciated activity in cancer cells [28]. However, stry of Higher Education (MOHE), Malaysia. CHST1 expression was induced in cancer pa- tients undergoing chemotherapy treatment and The first author would like to acknowledge the demonstrated protective effect on cells from support from the Ministry of Higher Education skin toxicity [53, 54]. In addition, “Zymosterol Malaysia for providing scholarship under the Biosynthesis” was modulated by down-regula- MyBrain (MyPhD) scheme. tion of TM7SF2. NT5E was down-regulated in both “Guanosine Nucleotides Degradation III” Disclosure of conflict of interest and “Urate Biosynthesis/Inosine 5’-phosphate Degradation” pathways. TM7SF2 is an enzyme None. that participates in the regulation of cholesterol homeostasis and inflammatory responses [55]. Address correspondence to: Rakesh Naidu, Jeffery Inhibition of TM7SF2 expression in skin papil- Cheah School of Medicine and Health Sciences, loma mouse showed reduced expression of Monash University Malaysia, Jalan Lagoon Selatan, genes associated with resistance to neoplastic 47500 Bandar Sunway, Selangor, Malaysia. E-mail: transformation and alteration in the expres- [email protected]

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Supplementary Table S1. Selected differentially expressed genes (DEGs) mutually regulated following 10 µM and 15 µM treatment of MS17 in PC-3 cells at 24 hrs Genbank Gene symbol Gene description 10 µM 15 µM p-Value GO term accession Up-regulation CRYAB* NM_001885 Crystallin, alpha B 277.6 537.6 5.42E-04 Cytoskeletal organization HSPA6* NM_002155 Heat shock 70 kDa protein 6 (HSP70B’) 145.2 336.7 1.09E-03 Response to unfolded protein MAFB NM_005461 V-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B 49.6 182.9 2.26E-03 Regulation of transcription RFPL2 NM_006605 Ret finger protein-like 2 49.2 128.8 3.23E-03 Binding activity NR1H4 NM_005123 Nuclear receptor subfamily 1, group H, member 4 41.6 88.5 5.77E-03 Regulation of transcription HSPB8* NM_014365 Heat shock 22 kDa protein 8 41.3 88.4 1.67E-03 Response to unfolded protein TRIML2* NM_173553 Tripartite motif family-like 2 30.2 88.1 6.54E-03 Protein ubiquitination HSPB2 NM_001541 Homo sapiens heat shock 27 kDa protein 2 (HSPB2), mRNA 28.7 77.9 6.17E-03 Response to unfolded protein KRTAP21-2 NM_181617 Keratin associated protein 21-2 27.9 56.8 1.48E-03 Cytoskeletal organization INSL4 NM_002195 Insulin-like 4 (placenta) 19.4 54.3 4.87E-03 Cell proliferation DNAI2* NM_023036 Dynein, axonemal, intermediate chain 2 24 51.9 7.84E-03 Cytoskeletal organization CACNA1G* NM_018896 Calcium channel, voltage-dependent, T type, alpha 1G subunit 17 48.3 2.51E-03 Transporter activity KRTAP3-2 NM_031959 Keratin associated protein 3-2 18.8 47.9 2.11E-02 Cytoskeletal organization FABP3 NM_004102 Fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) 19.4 41.2 2.44E-03 Transporter activity RFPL1 NM_021026 Ret finger protein-like 1 13.3 38.5 4.36E-03 Binding activity DAZ1 NM_004081 Deleted in azoospermia 1 12.4 37.2 1.38E-02 Reproductive process MMP3* NM_002422 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) 15.6 33.9 2.92E-03 Proteolysis SPANXB2 NM_145664 SPANX family, member B2 20.4 33.6 1.37E-03 Reproductive process IL10RA NM_001558 Interleukin 10 receptor, alpha 13.7 32.1 8.38E-03 Immune response DNAJA4* NM_018602 DnaJ (Hsp40) homolog, subfamily A, member 4 16.3 31.7 1.55E-03 Protein folding BATF NM_006399 Basic leucine zipper transcription factor, ATF-like 12.2 30.3 9.65E-03 Regulation of transcription GADD45G NM_006705 Growth arrest and DNA-damage-inducible, gamma 9.9 27 3.07E-03 Apoptosis SPANXA1 NM_013453 Sperm protein associated with the nucleus, X-linked, family member A1 19.2 27 1.23E-03 Reproductive process TRIM54 NM_032546 Tripartite motif containing 54 14.9 24.4 1.23E-03 Cytoskeletal organization HTR3C NM_130770 5-hydroxytryptamine (serotonin) receptor 3C, ionotropic 10 24.3 4.56E-03 Transporter activity NGFR* NM_002507 Nerve growth factor receptor 14.2 23.8 6.34E-03 Apoptosis CCL26* NM_006072 Chemokine (C-C motif) ligand 26 13 23.1 1.51E-03 Immune response DNAJB4 NM_007034 DnaJ (Hsp40) homolog, subfamily B, member 4 10.4 22.2 1.67E-03 Protein folding RRAD NM_004165 Ras-related associated with diabetes 8.8 21.4 3.42E-03 Signal transduction MMP10* NM_002425 Matrix metallopeptidase 10 (stromelysin 2) 9.2 20.9 6.03E-03 Proteolysis ABL2 NM_007314 C-abl oncogene 2, non-receptor tyrosine kinase 9.1 18.7 1.24E-03 Cytoskeletal organization HSPA1L NM_005527 Heat shock 70 kDa protein 1-like 6.7 18.4 3.21E-03 Response to unfolded protein LBH NM_030915 Limb bud and heart development 7.2 18.1 6.54E-03 Regulation of transcription Down-regulation CTDSP1* NM_021198 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 1 -5.8 -9.3 6.59E-04 Phosphatase activity MKI67 NM_002417 Marker of proliferation Ki-67 -3.5 -7.6 1.41E-03 Cell proliferation

1 Gene expression of prostate cancer cells

AURKB NM_004217 Aurora kinase B -2.7 -6.6 2.25E-03 Cell cycle HIST2H3A NM_001005464 Histone cluster 2, H3a -4.2 -6.5 9.56E-04 Chromosome organization NEIL3 NM_018248 Nei endonuclease VIII-like 3 (E. coli) -2.9 -6.5 2.69E-03 DNA repair TOP2A NM_001067 Topoisomerase (DNA) II alpha 170 kDa -2.5 -6.4 2.95E-03 DNA replication HLF NM_002126 Hepatic leukemia factor -3 -6.4 1.21E-02 Regulation of transcription CDCA3 NM_031299 Cell division cycle associated 3 -3 -5.8 1.48E-03 Cell cycle GTSE1 NM_016426 G-2 and S-phase expressed 1 -2.7 -5.8 1.63E-03 Cell cycle HIST1H1D NM_005320 Histone cluster 1, H1d -3.2 -5.7 1.29E-03 Chromosome organization HIST1H2AI* NM_003509 Histone cluster 1, H2ai -3.7 -5.7 1.66E-03 Chromosome organization PANX1 NM_015368 Pannexin 1 -3.5 -5.6 2.76E-03 Transporter activity KIF2C NM_006845 Kinesin family member 2C -2.5 -5.3 2.51E-03 Cytoskeletal organization KIF11 NM_004523 Kinesin family member 11 -3 -5.2 1.63E-03 Cytoskeletal organization E2F8 NM_024680 E2F transcription factor 8 -2.5 -5.1 4.62E-03 Regulation of transcription KIF20A NM_005733 Kinesin family member 20A -2.5 -5 2.06E-03 Cytoskeletal organization CDK1 NM_001786 Cyclin-dependent kinase 1 -2.6 -4.9 2.34E-03 Cell cycle DLGAP5 NM_014750 Discs, large (Drosophila) homolog-associated protein 5 -2.5 -4.9 2.91E-03 Cell proliferation HIST1H3B NM_003537 Histone cluster 1, H3b -3.3 -4.9 1.74E-03 Chromosome organization ASPM NM_018136 Asp (abnormal spindle) homolog, microcephaly associated (Drosophila) -2.6 -4.7 1.41E-03 Cell cycle FBXO5 NM_001142522 F-box protein 5 -2.8 -4.7 1.88E-03 Cell cycle HMMR NM_012484 Hyaluronan-mediated motility receptor -2.7 -4.6 2.63E-03 Binding activity CLDN11 NM_005602 Claudin 11 -3 -4.6 1.65E-03 Cell adhesion HIST1H2BF* NM_003522 Histone cluster 1, H2bf -3.5 -4.6 1.88E-03 Chromosome organization KIF15 NM_020242 Kinesin family member 15 -2.7 -4.6 1.58E-03 Cytoskeletal organization TROAP NM_005480 Trophinin associated protein -2.3 -4.4 1.41E-03 Cell adhesion SPC24 NM_182513 SPC24, NDC80 kinetochore complex component -2.7 -4.4 1.01E-03 Cell cycle BLM NM_000057 Bloom syndrome, RecQ helicase-like -2.4 -4.4 1.53E-03 DNA replication DEPDC1 NM_017779 DEP domain containing 1 -2.3 -4.4 4.54E-03 Signal transduction SLFN13 NM_144682 Schlafen family member 13 -3.6 -4.3 3.96E-03 Binding activity PLK1 NM_005030 Polo-like kinase 1 -2.3 -4.3 6.34E-03 Cell cycle KIFC1 NM_002263 Kinesin family member C1 -2.2 -4.3 4.47E-03 Cytoskeletal organization CCNB2 NM_004701 Cyclin B2 -2.4 -4.2 1.86E-03 Cell cycle HAPLN1 NM_001884 Hyaluronan and proteoglycan link protein 1 -3.2 -4.1 3.19E-02 Cell adhesion NCAPG NM_022346 Non-SMC condensin I complex, subunit G -2.6 -4.1 8.53E-03 Chromosome organization SLC14A1 NM_001146037 Solute carrier family 14 (urea transporter), member 1 (Kidd blood group) -2.7 -4.1 1.76E-03 Regulation of transcription TNFRSF6B* NM_003823 Tumor necrosis factor receptor superfamily, member 6b, decoy -3.4 -4 2.10E-02 Apoptosis TNS4 NM_032865 Tensin 4 -2.3 -4 2.62E-02 Apoptosis PRR11 NM_018304 Proline rich 11 -2.5 -4 3.17E-03 Cell cycle DNAH5 NM_001369 Dynein, axonemal, heavy chain 5 -2.6 -4 1.48E-03 Cytoskeletal organization NDC80 NM_006101 NDC80 kinetochore complex component -2.7 -4 1.81E-03 Cytoskeletal organization E2F2 NM_004091 E2F transcription factor 2 -2 -4 2.23E-03 Regulation of transcription CENPA NM_001809 Centromere protein A -2.3 -3.9 2.63E-03 Cell cycle

2 Gene expression of prostate cancer cells

MND1 NM_032117 Meiotic nuclear divisions 1 homolog (S. cerevisiae) -2.4 -3.9 3.16E-03 Cell cycle ASF1B NM_018154 Anti-silencing function 1B histone chaperone -2.3 -3.9 2.06E-03 Chromosome organization HIST2H2AC NM_003517 Histone cluster 2, H2ac -2.9 -3.9 1.63E-03 Chromosome organization CKAP2L NM_152515 Cytoskeleton associated protein 2-like -2.2 -3.9 7.08E-03 Cytoskeletal organization MMS22L NM_198468 MMS22-like, DNA repair protein -2.2 -3.9 2.91E-03 DNA replication BUB1B NM_001211 BUB1 mitotic checkpoint serine/threonine kinase B -2.3 -3.8 1.23E-03 Cell cycle CASC5 NM_170589 Cancer susceptibility candidate 5 -2.5 -3.8 2.08E-03 Chromosome organization POC1A NM_015426 POC1 centriolar protein A -2.3 -3.8 1.58E-03 Chromosome organization FANCA NM_000135 Fanconi anemia, complementation group A -2.4 -3.8 2.16E-03 DNA repair NMU NM_006681 Neuromedin U -2.5 -3.8 3.05E-03 Signal transduction RIBC2 NM_015653 RIB43A domain with coiled-coils 2 -2.1 -3.7 2.43E-03 Binding activity NUF2 NM_145697 NUF2, NDC80 kinetochore complex component -2.5 -3.7 3.03E-03 Chromosome organization MXD3* NM_031300 MAX dimerization protein 3 -2.6 -3.7 4.18E-03 Regulation of transcription *Green represents mutually up-regulated genes while red indicates mutually down-regulated genes in DU145 cells.

Supplementary Table S2. Selected differentially expressed genes (DEGs) mutually regulated following 10 µM and 15 µM treatment of MS17 in DU145 cells at 24 hrs Genbank Gene symbol Gene description 10 µM 15 µM p-Value GO term accession Up-regulation HSPA6* NM_002155 Heat shock 70 kDa protein 6 (HSP70B’) 373.4 814.4 4.96E-04 Response to unfolded protein CRYAB* NM_001885 Crystallin, alpha B 334.9 493.9 5.46E-04 Cytoskeletal organization MMP10* NM_002425 Matrix metallopeptidase 10 (stromelysin 2) 96.7 165.2 5.11E-03 Proteolysis MMP3* NM_002422 Matrix metallopeptidase 3 (stromelysin 1, progelatinase) 71.6 122.8 5.83E-03 Proteolysis CCL26* NM_006072 Chemokine (C-C motif) ligand 26 56 81.4 1.70E-03 Immune response CACNA1G* NM_018896 Calcium channel, voltage-dependent, T type, alpha 1G subunit 23 60.2 3.60E-02 Transporter activity NGFR* NM_002507 Nerve growth factor receptor 31.5 53.6 1.15E-02 Apoptosis DEFB103B NM_018661 Defensin, beta 103B 54.7 50.9 5.57E-04 Immune response ATF3 NM_001040619 Activating transcription factor 3 28.7 45.7 1.48E-03 Regulation of transcription MMP1 NM_002421 Matrix metallopeptidase 1 (interstitial collagenase) 37.3 43.1 1.32E-03 Proteolysis ZNF556 NM_024967 Zinc finger protein 556 22 41.3 1.46E-02 Regulation of transcription KRT14 NM_000526 Keratin 14 17.7 36.1 2.51E-03 Structural molecule activity DNAI2* NM_023036 Dynein, axonemal, intermediate chain 2 17.6 32.1 3.77E-02 Cytoskeletal organization NRAP NM_198060 Nebulin-related anchoring protein 15.2 29.5 3.60E-02 Binding activity KRT34 NM_021013 Keratin 34 17.8 25.4 5.36E-03 Structural molecule activity TRIML2* NM_173553 Tripartite motif family-like 2 11.6 24.7 3.61E-02 Protein ubiquitination PGF NM_002632 Placental growth factor 13.1 24 1.54E-02 Angiogenesis SDCBP2 NM_080489 Syndecan binding protein (syntenin) 2 14.3 21.6 4.40E-04 Binding activity RFPL4AL1 NM_001277397 Ret finger protein-like 4A-like 1 11 19.8 2.58E-02 Binding activity HEY1 NM_001040708 Hes-related family bHLH transcription factor with YRPW motif 1 9.3 19.8 4.48E-02 Regulation of transcription

3 Gene expression of prostate cancer cells

DNAJA4* NM_018602 DnaJ (Hsp40) homolog, subfamily A, member 4 11.5 19.1 3.87E-03 Protein folding CHST1 NM_003654 Carbohydrate (keratan sulfate Gal-6) sulfotransferase 1 11.4 18.8 2.23E-03 Metabolic process ZNF844 NM_001136501 Zinc finger protein 844 11.6 18 1.37E-02 Regulation of transcription HSPB8* NM_014365 Heat shock 22 kDa protein 8 12 18 2.89E-02 Response to unfolded protein SCN3B NM_018400 Sodium channel, voltage-gated, type III, beta subunit 9 17.6 3.74E-02 Transporter activity DHRS2 NM_182908 Dehydrogenase/reductase (SDR family) member 2 11.5 16.7 6.31E-03 Oxidoreductase activity ARHGEF7 NM_145735 Rho guanine nucleotide exchange factor (GEF) 7 10.4 16.7 1.44E-02 Signal transduction activity SERPIND1 NM_000185 Serpin peptidase inhibitor, clade D (heparin cofactor), member 1 11.6 16.5 2.10E-02 Endopeptidase activity AQP1 NM_198098 Aquaporin 1 (Colton blood group) 10.6 16.5 9.07E-03 Transporter activity SYNPO2L NM_024875 Synaptopodin 2-like 9 16.1 2.58E-02 Cytoskeletal organization GCGR NM_000160 Glucagon receptor 10.5 15.9 3.01E-02 Signal transduction activity MAL NM_002371 Mal, T-cell differentiation protein 7.5 15.8 3.18E-02 Apoptosis Down-regulation TNFRSF6B* NM_003823 Tumor necrosis factor receptor superfamily, member 6b, decoy -4.5 -6.5 3.66E-03 Apoptosis CTDSP1* NM_021198 CTD (carboxy-terminal domain, RNA polymerase II, polypeptide A) small phosphatase 1 -4.7 -5.8 6.10E-03 Phosphatase activity DGAT2 NM_032564 Diacylglycerol O-acyltransferase 2 -3.6 -4.6 1.16E-02 Metabolic process LHX3 NM_014564 LIM homeobox 3 -2.5 -4.5 6.56E-03 Regulation of transcription DTX4 NM_015177 Deltex homolog 4 (Drosophila) -4.1 -4.2 1.39E-03 Cell cycle B9D1 NM_015681 B9 protein domain 1 -2.9 -3.7 1.32E-03 Receptor activity HIST1H2BF* NM_003522 Histone cluster 1, H2bf -2.7 -3.3 2.38E-02 Chromosome organization CYP24A1 NM_000782 Cytochrome P450, family 24, subfamily A, polypeptide 1 -2.9 -3.3 2.07E-02 Oxidoreductase activity LOXL2 NM_002318 Lysyl oxidase-like 2 -2.7 -3.2 2.88E-02 Oxidoreductase activity PRKAG1 NM_001206710 Protein kinase, AMP-activated, gamma 1 non-catalytic subunit -2.4 -3.2 1.59E-02 Protein kinase activity RAB26 NM_014353 RAB26, member RAS oncogene family -2.4 -3.1 1.53E-02 Signal transduction activity MPZL2 NM_144765 Myelin protein zero-like 2 -2.3 -3 1.59E-02 Cell adhesion PARD6A NM_016948 Par-6 family cell polarity regulator alpha -2.5 -3 4.28E-02 Cell cycle PHYHD1 NM_174933 Phytanoyl-CoA dioxygenase domain containing 1 -2.3 -3 1.97E-02 Oxidoreductase activity MAP2K6 NM_002758 Mitogen-activated protein kinase kinase 6 -2.4 -3 7.31E-03 Protein kinase activity ARHGEF9 NM_015185 Cdc42 guanine nucleotide exchange factor (GEF) 9 -2.4 -3 6.02E-03 Signal transduction activity HOXC4 NM_014620 Homeobox protein Hox-C4 -2.7 -2.9 8.29E-03 Regulation of transcription STMN3 NM_015894 Stathmin-like 3 -2.5 -2.8 2.91E-02 Binding activity HIST1H2AI* NM_003509 Histone cluster 1, H2ai -2.6 -2.8 3.15E-03 Chromosome organization B3GNT4 NM_030765 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 4 -2.1 -2.8 3.52E-02 Metabolic process GABRD NM_000815 Gamma-aminobutyric acid (GABA) A receptor, delta -2.3 -2.8 3.86E-03 Signal transduction activity TBCD NM_005993 Tubulin folding cofactor D -2.3 -2.7 2.95E-02 Binding activity CLEC11A NM_002975 C-type lectin domain family 11, member A -2.2 -2.7 2.45E-02 Cell proliferation PADI2 NM_007365 Peptidyl arginine deiminase, type II -2.6 -2.7 1.44E-02 Chromosome organization PTGDS NM_000954 Prostaglandin D2 synthase 21 kDa (brain) -2.3 -2.7 1.10E-02 Metabolic process NT5E NM_002526 5’-nucleotidase, ecto (CD73) -2 -2.7 3.94E-04 Metabolic process ALDH3A1 NM_001135168 Aldehyde dehydrogenase 3 family, member A1 -2 -2.7 3.23E-02 Oxidoreductase activity PAQR4 NM_152341 Progestin and adipoQ receptor family member IV -2.4 -2.7 2.84E-03 Receptor activity

4 Gene expression of prostate cancer cells

MXD3* NM_031300 MAX dimerization protein 3 -2 -2.7 1.02E-02 Regulation of transcription WWC3 NM_015691 WWC family member 3 -2.1 -2.7 5.95E-03 Regulation of transcription NAT14 NM_020378 N-acetyltransferase 14 (GCN5-related, putative) -2.3 -2.7 1.10E-02 Transferase activity MTFP1 NM_016498 Mitochondrial fission process 1 -2.7 -2.6 2.58E-02 Apoptosis EGFL7 NM_201446 EGF like Domain Multiple 7 -2.4 -2.6 3.86E-04 Binding activity CCDC80 NM_199511 Coiled-coil domain containing 80 -2 -2.6 5.32E-03 Binding activity LTBP2 NM_000428 Latent transforming growth factor beta binding protein 2 -2.1 -2.6 2.47E-02 Cell adhesion TM7SF2 NM_003273 Transmembrane 7 superfamily member 2 -2.2 -2.6 1.42E-03 Metabolic process GRHPR NM_012203 Glyoxylate reductase/hydroxypyruvate reductase -2.3 -2.6 2.28E-03 Oxidoreductase activity DHRS4 NM_001282989 Dehydrogenase/reductase (SDR family) member 4 -2.2 -2.6 2.97E-02 Oxidoreductase activity MAMDC4 NM_206920 MAM domain containing 4 -2.3 -2.6 4.69E-03 Transporter activity SYTL2 NM_206927 Synaptotagmin-like 2 -2.2 -2.6 3.51E-02 Transporter activity KCNMA1 NM_002247 Potassium large conductance calcium-activated channel, subfamily M, alpha member 1 -2.1 -2.6 1.19E-02 Transporter activity NCKIPSD NM_016453 NCK interacting protein with SH3 domain -2.1 -2.5 1.52E-02 Binding activity NUDT8 NM_001243750 Nudix (nucleoside diphosphate linked moiety X)-type motif 8 -2.2 -2.5 8.33E-03 Metabolic process LOXL1 NM_005576 Lysyl oxidase-like 1 -2.3 -2.5 1.16E-03 Oxidoreductase activity LY9 NM_001033667 Lymphocyte antigen 9 -2 -2.5 3.55E-03 Protein kinase activity SFRP1 NM_003012 Secreted frizzled-related protein 1 -2.1 -2.5 6.84E-03 Receptor activity SPDEF NM_012391 SAM pointed domain containing ETS transcription factor -2.2 -2.5 1.14E-02 Regulation of transcription ARHGDIB NM_001175 Rho GDP dissociation inhibitor (GDI) beta -2.2 -2.5 2.61E-03 Signal transduction activity SLC27A5 NM_012254 Solute carrier family 27 (fatty acid transporter), member 5 -2.2 -2.5 6.01E-04 Transporter activity *Green represents mutually up-regulated genes while red indicates mutually down-regulated genes in PC-3 cells.

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